In systemic sclerosis (SSc), excessive accumulation of collagen leads to fibrosis and subsequent dysfunction of skin and internal organs. Transforming growth factor-beta (TGF-beta) plays a pivotal role in initiating and propagating fibrosis. Because of its key role in SSc, understanding how TGF-beta regulates fibroblast functions would clarify the pathogenesis of SSc and identify novel targets for therapeutic intervention. The recent discovery of the Smad family of intracellular TGF-beta signal transducers and the recognition of their major roles in multiple diseases, provides novel approaches for investigating the involvement of TGF-beta in pathogenesis of SSc. The proposed studies utilize a combination of in vitro and in vivo approaches in order to answer four important questions fundamental to understanding the role of the TGF-beta/Smad axis in SSc: What are fibrosis-related genes whose expression in human fibroblasts is modulated by Smad3? How is the Smad pathway integrated into the cellular signaling network of normal and SSc fibroblasts during inflammation and repair? Are intrinsic alterations in Smad signaling implicated in the SSc fibroblast phenotype? What is the functional role of Smad3 in the development of scleroderma in mice? Our hypothesis is that fibrosis in SSc is due to sustained activation of fibroblast gene expression, and that Smad3 plays a pivotal role in this process. The hypothesis will be evaluated in four interrelated aims. In Specific Aim 1, we will use DNA microarrays to profile genes whose expression is modulated by inducible expression of Smad3 in immortalized human fibroblasts; In Specific Aim 2, we will examine modulation of fibroblast TGF-beta/Smad signaling by inflammatory cells through CD4O. In Specific Aim 3, we will characterize the Smad pathway in SSc fibroblasts and tissues in order to identify intrinsic alterations in the regulation and/or function of the TGF-beta/Smad axis in SSc that may contribute to fibroblast activation. Observations derived from in vitro experiments cannot always accurately predict protein function in vivo. We hypothesize that Smad3 is an essential mediator of TGF-beta signaling, and mice lacking Smad3 have reduced susceptibility to pathological tissue fibrosis. To test this hypothesis directly, in Specific Aim 4 we will induce cutaneous fibrosis in Smad3-null mice in order to determine the role of Smad3 in this process. The results will clarify the role of Smad signal pathway in mediating the development of fibrosis in SSc, and reveal the feasibility of therapeutically targeting.